Potentiation of Glycine Receptors by Ethanol

乙醇对甘氨酸受体的增强作用

• 批准号: 7660486
• 负责人: LUIS GERARDO AGUAYO
• 金额: $ 16.4万
• 依托单位: UNIVERSITY OF CONCEPCION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660486
• 关键词: Affect; Agreement; Alcohol consumption; Alcoholic Intoxication; Alcohols; Amino Acid Motifs; Amino Acids; Attenuated; Basic Amino Acids; Binding; Biochemical; Brain; Brain Stem; Buffers; Calcium Channel; Cell membrane; Cessation of life; Chemosensitization; Complement; Complex; Consumption; Convulsions; Data; Development; Ethanol; Face; G Beta Gamma; GTP Binding; GTP-Binding Proteins; General anesthetic drugs; Glycine; Glycine Receptors; Hand; Health; Human; Isoflurane; Kinetics; Laboratories; Modification; Molecular; Muscle Tonus; Muscle relaxation phase; Mutagenesis; Mutate; Mutation; Nature; Neuraxis; Neurologic; Neurons; Pain; Peptides; Pharmaceutical Preparations; Phenotype; Physiological; Play; Property; Propofol; Protein Subunits; Rattus; Research; Resistance; Respiration; Role; Scanning; Sedation procedure; Sensory; Series; Site; Spinal Cord; System; Techniques; Testing; Toxin; Western Blotting; Work; alcohol effect; alcohol sensitivity; alcoholism therapy; base; chemical property; clinically relevant; dimer; motor control; mutant; neuronal excitability; novel; patch clamp; protein activation; public health relevance; receptor; receptor binding; receptor sensitivity; research study; respiratory; sensory integration; stoichiometry; voltage

DESCRIPTION (provided by applicant): Activation of glycine receptors (GlyRs) in brain stem, spinal cord and brain provides a main inhibitory control for neuronal excitability. Modification of these receptors by toxins and drugs such as ethanol leads to a wide range of effects varying from sedation and muscle relaxation to convulsions, respiratory arrest and death. However, the mechanism by which ethanol affects these receptors is largely unknown. Recent studies have indicated that GlyR function is modified by changes in GTP-binding protein activity. Specifically, using a series of GlyR mutants, the presence of basic amino acid motifs that are responsible for G beta gamma binding and receptor modulation were discovered. More importantly, preliminary results show that basic amino acids in the main intracellular loop of the GlyR determine the sensitivity of the receptor to clinically relevant concentrations of ethanol (10-100 mM). These amino acids also regulate the interaction of the GlyR with G beta gamma. In agreement with these findings, it was found that GlyR sensitivity to ethanol was significantly modified by changing the stoichiometry of the heterotrimeric complex. Therefore, it was hypothesized that important determinants for ethanol sensitivity are within the intracellular loop and that the sensitivity of GlyR to ethanol depends on the state of G protein activation. Remarkably, it was shown that these sites did not participate in the modulation of GlyRs by propofol, isoflurane and long chain alcohols indicating a novel and selective new site for ethanol. Additionally, mutations in these sites did not affect the physiological properties of the GlyR indicating that the ethanol resistant phenotype was not related to changes on channel gating. Therefore, the primary objective of this study is to characterize the intracellular sites responsible for G protein activation and GlyR sensitivity to ethanol by using receptor mutagenesis, kinetic analysis of GTP binding to G proteins, heterologous expression of G protein subunits, patch-clamp and Western blot techniques. In addition, key experiments will be performed using WT and mutant GlyRs expressed in a neuronal background (i.e. DRG neurons). The information obtained from this study will help to understand the mechanism by which ethanol affects glycine receptors, which are important in functions such as convulsions, sensory integration, muscle tone and respiration. Therefore, these results may contribute to the development of new treatments for alcoholism. PUBLIC HEALTH RELEVANCE Excessive consumption of ethanol is a main health-related problem in the US and world wide. However, the main determinants for the changes in the brain functions produced by ethanol are still largely unknown. Therefore, it is important to identify the mechanism and sites of ethanol action in order to understand how ethanol affects the central nervous system and to propose useful therapies to alleviate neurological health problems associated with its excessive consumption.

描述(由申请人提供)：激活脑干、脊髓和大脑中的甘氨酸受体(GlyRs)为神经元兴奋性提供了主要的抑制控制。用毒素和药物(如乙醇)修饰这些受体会导致从镇静和肌肉松弛到抽搐、呼吸停止和死亡等一系列不同的效果。然而，乙醇影响这些受体的机制在很大程度上是未知的。最近的研究表明，GlyR的功能受到GTP结合蛋白活性变化的影响。具体地说，使用一系列GlyR突变体，发现了负责Gβ伽马结合和受体调节的碱性氨基酸基序的存在。更重要的是，初步结果表明，GlyR主要细胞内环中的碱性氨基酸决定了受体对临床相关浓度(10-100 mM)乙醇的敏感性。这些氨基酸还调节GlyR与Gβ伽马的相互作用。与这些发现一致的是，研究发现，通过改变杂三聚体络合物的化学计量比，GlyR对乙醇的敏感性得到了显著改善。因此，假设乙醇敏感性的重要决定因素在细胞内环内，GlyR对乙醇的敏感性取决于G蛋白的激活状态。值得注意的是，这些位点不参与异丙酚、异氟醚和长链醇对GlyRs的调节，这表明乙醇是一个新的、选择性的新位点。此外，这些位点的突变不影响GlyR的生理特性，表明乙醇抗性表型与通道门控的变化无关。因此，本研究的主要目的是通过受体突变、GTP与G蛋白结合的动力学分析、G蛋白亚基的异源表达、膜片钳技术和Western印迹技术来鉴定导致G蛋白激活和GlyR对乙醇敏感性的细胞内位点。此外，关键实验将使用WT和突变型GlyRs在神经元背景(即DRG神经元)中表达。从这项研究中获得的信息将有助于理解乙醇影响甘氨酸受体的机制，甘氨酸受体在抽搐、感觉整合、肌肉紧张性和呼吸等功能中非常重要。因此，这些结果可能有助于开发新的酒精中毒治疗方法。在美国和世界范围内，乙醇的过度消费是一个与健康相关的主要问题。然而，乙醇引起的大脑功能变化的主要决定因素在很大程度上仍然未知。因此，为了了解乙醇如何影响中枢神经系统，并提出有效的治疗方法来缓解与其过度摄入相关的神经健康问题，确定乙醇的作用机制和部位是很重要的。